Right-handed Z-DNA at ultrahigh resolution: a tale of two hands and the power of the crystallographic method

The crystal structure of the self-complementary d(CGCGCG)2 Z-DNA duplex in complex with cadaverinium and potassium cations was solved at ultrahigh resolution. The oligonucleotide used for crystallization contained the enantiomeric 2′-deoxy-l-ribose instead of its natural d-enantiomer, and thus the Z-DNA duplex is right-handed.


Introduction
Natural d-deoxyoligonucleotides [for example d-d(CGCGCG) 2 ; d-DNA] and their synthetic enantiomers [l-d(CGCGCG) 2 , l-DNA] with the same sequence have the same physical (for example duplex stability or solubility) and chemical properties (Hauser et al., 2006). However, the l-enantiomers do not interact with biological partners and, not surprisingly, they cannot serve as templates for DNA or RNA polymerases (Hayashi et al., 2005;Hauser et al., 2006). The l-enantiomers are also resistant to nuclease degradation and to many offtarget interactions that plague traditional d-oligonucleotidebased technologies (Anderson et al., 1984;Asseline et al., 1991;Damha et al., 1994;Williams et al., 1997;Hauser et al., 2006). This makes them ideal for biomedical applications (for example as aptamers or biosensors), as well as attractive objects for molecular biology (for example for chiral separations) or DNA nanotechnology (Young et al., 2019).
It has been shown that polyamines are able to induce secondary-structure transitions of DNA, with the B-Z transition of alternating purine/pyrimidine oligonucleotides in solution being particularly enhanced by polyamines (van Dam et al., 2002). Cadaverine 2+ [Cad 2+ ; þ H 3 NðCH 2 Þ 5 NH þ 3 ], a biogenic amine, is a positively charged organic dication under physiological ionic and pH conditions and hence can interact with negatively charged macromolecules such as DNA and RNA. In solution, 1,3-diaminopropane (DAP) and diaminoethane are similarly effective in the B-Z conversion, with both being slightly more effective than putrescine 2+ [Put 2+ ; þ H 3 NðCH 2 Þ 4 NH þ 3 ] and more effective than Cad 2+ (Behe & Felsenfeld, 1981). In another study, thermodynamic analysis of the interactions of biogenic polyamines with genomic DNA showed that polyamines bound more strongly to AT-rich DNA compared with DNA with a high GC content and that the binding efficiency varied depending on the charge of the polyamine as follows: spermine 4+ > spermidine 3+ > putrescine 2+ > cadaverine 2+ (Kabir & Kumar, 2013). However, the Cad 2+ ion is specified as a ligand in only a few protein structures deposited in the PDB (Berman et al., 2000). Thus, Cad 2+ is very poorly characterized in the context of its interactions with nucleic acids.
In this paper, we describe an ultrahigh-resolution crystal structure of Z-DNA with the sequence l-d(CGCGCG) 2 , in which the natural 2 0 -deoxy-d-ribose was replaced with its l-enantiomer. This is the first crystal structure determination of right-handed Z-DNA, with the caveat that the right-handed enantiomer was also present, together with the natural lefthanded duplex, in the centrosymmetric crystal structure of the same DNA (Doi et al., 1993;Drozdzal et al., 2016).
The story behind this project is very interesting and demonstrates the power and sensitivity of the crystallographic method. The postdoc who was synthesizing the d(CGCGCG) 2 duplex for a crystallization experiment with cadaverine 2+ ran out of the 2 0 -deoxy-d-ribose oligonucleotide, and thinking (basically correctly, if handedness is neglected) that it would not matter very much, used for this purpose the 2 0 -deoxy-lribose oligonucleotide at hand, a leftover from a 'centrosymmetric project' . He grew excellent crystals and then left. The ultrahigh-resolution X-ray diffraction data, obviously collected at a very short wavelength (0.7085 Å ), were inherited by another postdoc, who was unaware of the swap of hands in the crystallization experiment. He was extremely puzzled when his high-resolution refinements kept telling him that the model was wrong and that the structure enantiomorph should be inverted. This indication was given by the Flack and Pearson tests (Flack, 1983;Flack & Bernardinelli, 2008), as included in the SHELXL (Sheldrick, 2015) software, and also by the Hooft test (Hooft et al., 2008) as implemented in PLATON (Spek, 2009). After switching the structure enantiomorph the refinement was unproblematic, but the Z-DNA duplex became right-handed. The first postdoc was then contacted and he explained his experiment. At this point everything started making sense again.
In addition to describing an ultrahigh-resolution structure of a right-handed Z-DNA duplex, this paper also provides, for the first time, insight into the interaction of (Z-)DNA with the cadaverinium dication (Cad 2+ ). After inspection of all of the crystal structures of Z-form DNA in the PDB as well as those described in the scientific literature, we compiled a list of 68 nucleic acid structures in complex with polyamines and/or metal cations (Supplementary Table S1). Among these crystal structures, there are 44 Z-DNA/metal m+ complexes, 16 Z-DNA/polyamine n+ /metal m+ and eight Z-DNA/polyamine n+ complexes, none them with cadaverine.

Oligonucleotide synthesis, purification and crystallization
The methods for the synthesis, deprotection and purification of the oligodeoxynucleotide have been described previously (Xia et al., 1998;Drozdzal et al., 2013). A 1.5 mM water solution of the 2 0 -deoxy-l-ribose-containing DNA oligonucleotide with the self-complementary sequence l-d(CGCGCG) was heated at 338 K for 10 min and then slowly annealed to room temperature overnight. Single crystals of l-d(CGCGCG) 2 /Cad 2+ /K + were grown at 292 K by the hanging-drop vapor-diffusion method by mixing 2 ml nucleic acid solution and 2 ml precipitating solution consisting of 10%(v/v) (AE)-2-methyl-2,4-pentanediol (MPD), 40 mM sodium cacodylate pH 6.0, 80 mM KCl, 12 mM NaCl, 14 mM cadaverinium dichloride. The drops were equilibrated against 0.5 ml 80%(v/v) MPD. Crystals appeared within one week and grew to dimensions of 0.3 Â 0.1 Â 0.1 mm.

X-ray data collection and processing
X-ray diffraction data for the l-d(CGCGCG) 2 /Cad 2+ /K + complex were measured to 0.69 Å resolution on the EMBL P13 beamline at the PETRA III synchrotron at DESY, Hamburg. The crystal was vitrified in a stream of cold nitrogen gas at 100 K. The mother liquor served as a cryoprotectant. The diffraction data were collected at a wavelength of 0.7085 Å and were indexed, integrated and scaled using the XDS package (Kabsch, 2010), as summarized in Table 1.

Structure solution and refinement
The structure was solved by molecular replacement using Phaser (McCoy et al., 2007). The postdoc performing the structure-solution step was unaware that the crystal contained the l-d(CGCGCG) 2 'spiegelmer' of the oligonucleotide and he used, quite naturally, the DNA part of PDB entry 7atg, corresponding to our earlier model of the d-d(CGCGCG) 2 / Put 2+ /K + complex (Drozdzal et al., 2021), as the molecular probe. Since Friedel's law makes the diffraction pattern centrosymmetric (with the exception of the, usually small, deviations caused by anomalous scattering), a noncentrosymmetric crystal structure can be solved equally well, of course, by both enantiomers of the molecular model.
In the initial stages of the refinement, the model was refined using REFMAC5  from the CCP4 suite . The final anisotropic refinement was carried out with SHELXL (Sheldrick, 2015) using the full resolution of the diffraction data. The details of the SHELXL refinement were the same as described for our previous Z-DNA structures (Drozdzal et al., 2013(Drozdzal et al., , 2015. At this resolution, no stereochemical restraints are necessary to supplement the experimental observations (Jaskolski, 2017). However, restraints may still be needed for some disordered research papers or highly mobile fragments. In the present structure, restraints were only applied to the cadaverinium dication and to the bonds and angles of dual-conformation Z-DNA fragments. The ideal geometry targets for Cad 2+ were taken from a high-quality X-ray structure of cadaverinium dichloride (Pospieszna-Markiewicz et al., 2006). Conformation-dependent geometrical restraints on bond lengths (DFIX) and bond angles (DANG) for the polynucleotide chains were generated using the RestraintLib server (http://achesym.ibch.poznan.pl/ restraintlib/) as described by Kowiel et al. (2016Kowiel et al. ( , 2020 and Gilski et al. (2019). The CSD-derived conformation-dependent RestraintLib dictionary supersedes the classic nucleic acid restraints compiled by Clowney et al. (1996), Gelbin et al. (1996) and Parkinson et al. (1996). The final cycles of CGLS (conjugate-gradient least-squares) refinement converged with an R and R free of 10.32% and 12.83%, respectively. The very last round of refinement, calculated with the test reflections included in the working set, converged with R = 10.39%. In order to provide estimations of standard uncertainties in all individual refined parameters and all derived geometrical parameters, in the final stage of the refinement one cycle of full-matrix least-squares minimization was calculated. The placement of the model in the unit cell was standardized using the ACHESYM server (Kowiel et al., 2014).
At the wavelength used in the diffraction experiment (0.7085 Å ), the imaginary components of the anomalous scattering (f 00 ) of K and P atoms are 0.252 and 0.098 electron units, respectively (Cromer, 1983). Anomalous signal is visible in the diffraction data up to $0.9 Å resolution and therefore the refinement was carried out against unmerged anomalous data. The signal was quite weak, however, as no clear peaks were located in the anomalous electron-density map contoured at the 3 level that could correspond to the positions of the K + ion and P atoms.
Coot (Emsley et al., 2010) was used for visualization of the electron-density maps and manual rebuilding of the atomic model.

Determination of the absolute configuration of the d(CGCGCG) 2 DNA hexamer
Intriguingly, at this stage the structure refinement performed with SHELXL (Sheldrick, 2015) indicated an unexplained issue with model chirality. The Flack and Pearson parameters (Flack, 1983;Flack & Bernardinelli, 2008) calculated by the SHELXL program were 0.90 (8) and 0.95 (3), respectively. Moreover, model validation performed with PLATON (Spek, 2009) gave a similar warning, as the Flack, Pearson and Hooft (Hooft et al., 2008) parameters were 0.95 (3), 0.80 (2) and 0.89 (2), respectively. These results attracted our attention, as they obviously indicated the wrong configuration of the refined model. When it became clear that 2 0 -deoxy-l-ribose derivatives had been used instead of their natural d-counterparts in the synthesis of the deoxyoligonucleotide, the absolute configuration of the structure was inverted through the use of the MOVE command in SHELXL. When the inverted model was checked again with PLATON, the Flack, Pearson and Hooft parameters were calculated to be 0.03 (3), 0.12 (2) and 0.08 (2), respectively, clearly indicating the correct handedness, which corresponds to the right-handed l-d(CGCGCG) 2 DNA duplex. Also, the Flack and Pearson parameters calculated by the SHELXL program [0.03 (8)    jF obs j À jF calc j = P hkl jF obs j, where F obs and F calc are the observed and calculated structure factors, respectively. R free was calculated analogously for the test reflections, which were randomly selected and excluded from the refinement. covalent bonds are $0.006, $0.006, $0.005 and $0.004 Å for C-C, C-O, C-N and P-O, respectively. The r.m.s.d. agreement with stereochemical standards is 0.012 Å for bond lengths and 2.10 for bond angles.
The geometry of the duplex agrees well with the expected stereochemistry and with other crystallographic models of Z-DNA, including the ultrahigh-resolution PDB entry 3p4j (Supplementary Table S2) determined at 0.55 Å resolution (Brzezinski et al., 2011). The pseudorotation of the deoxyribose moieties, analyzed according to the method of Jaskó lski (1984), is typical for Z-DNA models and corresponds to the C2 0 -endo/C3 0 -endo pucker of the pyrimidine/purine 3 0 ,5 0nucleotides. Also in the present complex, the sugars at the 3 0 -termini do not have the alternating C2 0 -endo/C3 0 -endo pucker for the pyrimidine/purine nucleotides, as is typical for Z-DNA, but all assume the C2 0 -endo conformation. There are two conformational subforms of Z-DNA, namely ZI and ZII, differing in the torsion angles [defined as O3 0 (i À 1)-P-O5 0 -C5 0 ] and [C3 0 -O3 0 -P(i + 1)-O5 0 (i + 1)] (Saenger, 1984), stabilized by hydrogen bonding of a phosphate group to a hydrated metal cation or water molecule(s). Here, the ZII conformation of the phosphate group can only be assigned to G4(I), with = 62.7 (the signed torsion angle is given for the reference 2 0 -deoxy-d-ribose enantiomer). This alternative ZII conformer is stabilized by hydrogen bonds to the N1 atom of Cad 2+ and a water molecule. The remaining phosphate groups have the ZI conformation.

Binding of the cadaverinium dication
The entire cadaverinium dication is well defined in the electron-density map (Fig. 2) despite its partial occupancy of 0.526 (13). It has the all-trans conformation, with the following torsion angles: 170 (1), À173 (1), 172 (1) and 175 (2) . The Cad 2+ dication is only involved in hydrogen-bonding interactions with the O atoms of the phosphate groups of one DNA molecule ( Figs. 1 and 2). The N1 atom of the Cad 2+ dication forms hydrogen bonds to phosphate groups of the major (I) and minor (II) conformer. These include interactions with OP1(I)_G4 and OP1(II)_G4 at distances of 2.776 (22)  Overall structure of l-d(CGCGCG) 2 /Cad 2+ /K + highlighting the interactions between the cations and the DNA molecules within the asymmetric unit (orange) and two other symmetry-related DNA molecules (light orange): i (Àx, y + 1/2, Àz + 1/2) and ii (x + 1/2, Ày + 1/2, Àz). Potential polar interactions with the Cad 2+ cation (stick model) are marked as red dashed lines and K + is shown as a purple sphere with the coordination bonds marked as black dashed lines.

Figure 2
Details of the binding mode of the cadaverinium dication within the crystal lattice of the l-d(CGCGCG) 2 /Cad 2+ /K + complex. The 2mF o À DF c map (blue) is contoured at the 1.0 level. The color code for molecules and ions is the same as in Fig. 1. 2.833 (34) Å , respectively. The N2 atom is hydrogen-bonded to OP2(I)_C5 and OP1(II)_C5 at 2.760 (15) and 2.842 (15) Å , respectively. Additionally, the Cad 2+ ion forms hydrogen bonds to water molecules, which in turn interact with other phosphate groups of two symmetry-related molecules, indicated as i (Àx, y + 1/2, Àz + 1/2) and ii (x + 1/2, Ày + 1/2, Àz). It is of note that the Cad 2+ cation does not form any direct hydrogen bonds to symmetry-related Z-DNA molecules. We point out that since cadaverine is an achiral molecule, its interactions with both enantiomers of the Z-DNA molecule will be the same.

Coordination of the K + cation
The electron-density maps clearly revealed one metal coordination site, initially interpreted as K + (Fig. 3). Since the occupancy of this monovalent cation refined to a fractional value of 0.553 (9), a complementary water molecule was also modeled in the 2mF o À DF c map at this site. After refinement of this model, the occupancies of K + and the water molecule were 0.318 (17) and 0.682 (17), respectively. There are seven O atoms in the immediate vicinity of this site, four from the Z-DNA backbone (three OP atoms of two symmetry-related Z-DNA duplexes and one O5 0 hydroxyl O atom) and three from water molecules W4, W11 and W107 (the latter water molecule is partially disordered and has been modeled in two distinct positions). As in the previously studied Z-DNA/Put 2+ / K + complex (Drozdzal et al., 2021), the K + cation in the present structure is also located between two Z-DNA phosphate groups [OP1(II)_G6 and OP1/2(II)_C9 i ]. The K + -O bond distances are in the range 2.491 (12) to 3.017 (33) Å . The lengths of the M + -O bonds support the presence of K + rather than the presence of Na + at higher occupancy. The Check-MyMetal server (Zheng et al., 2014) also predicted potassium as the most likely cation at this site. The coordination sphere (coordination number seven) can be considered as distorted pentagonal bipyramid or a capped octahedron. The angles within the coordination sphere are irregular (Table 2).

Solvent structure and hydration of the right-handed Z-DNA helix
The crystallographic model presented in this work is similar to other high-resolution Z-DNA structures, also from the point of view of the architecture of the hydration shells (Drozdzal et al., 2013(Drozdzal et al., , 2015(Drozdzal et al., , 2021. Specifically, the asymmetric unit contains complicated hydrogen-bonded networks involved in crystal packing and stabilization of the conformation of the oligonucleotides. Not surprisingly, there are no significant differences in the hydration of the enantiomeric forms of Z-DNA. The asymmetric unit contains 121 water sites, which were refined anisotropically without positional restraints. There was no attempt to model the H atoms of the water molecules. There are 29 close pairs of water molecules with a combined occupancy of 1.0. The remaining water sites were classified as fully (25)  Potassium (purple sphere) coordination site in the crystal structure of the l-d(CGCGCG) 2 /Cad 2+ /K + complex. Water molecules (W) are presented as red spheres. The coordination sphere of this intermolecular potassium binding site is completed by partially disordered phosphate groups from two symmetry-related DNA molecules (orange and light orange). The 2mF o À DF c map (blue) is contoured at the 1.0 level. Table 2 Coordination geometry around the metal ion in the l-d(CGCGCG) 2 /Cad 2+ /K + structure.
Standard uncertainties are given in parentheses.

Distances (Å )
Angles (  of electrostatic neutrality of the crystal structure. The ten negatively charged phosphate groups are only partially neutralized by the two protonated amine groups of the partially occupied Cad 2+ dication and by the fractional potassium cation. Our attempts to identify other positively charged species within the asymmetric unit that would ensure electrostatic neutrality were unsuccessful. A similar problem was also unresolved even for the ultrahigh-resolution Z-DNA structure at 0.55 Å resolution (Brzezinski et al., 2011).

Discussion
In this work, we have presented a new crystal structure of right-handed Z-DNA in complex with cadaverine 2+ and K + cations. It is the first example of interaction of cadaverine 2+ with a DNA molecule. Since many functional l-DNA constructs are beginning to emerge, structural elucidation of l-DNA isomers and their complexes with biomacromolecules at the atomic level will be important for understanding how mirror DNA can be integrated into biological systems . Comparison of the positions of the potassium cations in the structures of left-handed Z-DNA with Put 2+ and of right-handed Z-DNA with Cad 2+ shows that despite the different chirality of the Z-DNA duplexes, the K + ion has a preference for binding at OP_G6. Our studies of the interactions of Z-DNA with metal and biogenic polyamine cations confirm the notion that the 'ionic atmosphere' created by these positively charged species not only plays an important role in neutralizing the net charge of the nucleic acid, but also affects the structural stability of the DNA (Xiao et al., 2014). It is remarkable that in four of the five metal complexes studied by us, the lability of the DNA molecules manifested itself as multiple conformations of the phosphate groups and even as double conformations of the bases (Zn 2+ , Cr 3+ , Mg 2+ and K + complexes; Drozdzal et al., 2013Drozdzal et al., , 2015Drozdzal et al., , 2016Drozdzal et al., , 2021. The above studies support the widely held opinion that from a chemical point of view, nucleic acid crystals are actually salts (or complexes) of metal ions. Thus, it is impossible to separate the behavior of these macromolecules from their interactions with metal cations (Kazakov & Hecht, 2006). In addition, double conformations of Z-DNA duplexes, often correlated with the presence of metals, may be important in mediating nucleic acid-protein interactions. To date, specific interactions of left-handed DNA with the Z domain of six binding proteins have been reported (Bartas et al., 2022). Analysis of the torsion angles of the Cad 2+ dication in this high-resolution structure, as well as in high-resolution structures of protein complexes with cadaverine (PDB entries 3qj5, 4ofg and 6ye7), indicates that in biological systems this simple diamine can adopt diverse conformations and consequently enter into a variety of interactions with biomacromolecular partners. Although the stabilization of nucleic acids by polyamine n+ cations has been studied for many years (Hou et al., 2001), the structures that we have presented so far mostly show that the interaction of biogenic polyamine polycations with DNA is not entirely synonymous with the absence of alternate conformations of the DNA sugar-phosphate back-bone. It was noted early on that the distances between the cationic centers of biogenic polyamines correspond well to the distances between the phosphate groups in DNA (Tsuboi, 1964;van Dam et al., 2002). As illustrated by our structure, this assumption is especially true for Cad 2+ , where the N1 + Á Á ÁN2 + distance of 7.451 (23) Å allows the formation of four hydrogen bonds to adjacent phosphate groups of the Z-DNA duplex (in the ideal Z-DNA hexamer, the PÁ Á ÁP distances are in the range $5.69 to $7.18 Å ). However, given the ability of polyamine n+ cations to adopt different conformations depending on the chemical environment in the crystal structures, it is reasonable to conclude that for both putrescine 2+ and cadaverine 2+ their (Z-)DNA binding site could change depending on the ionic conditions of the crystallization solution.
It is worth mentioning that not only mirror-image nucleotides can be used for the formation of right-handed Z-DNA. Satange et al. (2019) showed that the drug actinomycin D can tightly bind to G:G mismatched DNA duplexes through largescale structural rearrangements, resulting in a right-handed Z-DNA-type structure (PDB entry 6j0h). It could be interesting to investigate whether other drugs (antibiotics) or ligands could cause similar distortions of nucleic acid fragments.
Finally, the picturesque story of the present refinement underlies the notion that high-quality diffraction data do not lie, and that by the thorough and accurate observance of stateof-the-art crystallographic practices one can even detect and rectify potential mishaps related to poor communication or to project transfer.

Data availability
The atomic coordinates and anisotropic ADPs, as well as the processed structure factors corresponding to the final model presented in this work, have been deposited in the PDB with accession code 8a71. The raw X-ray diffraction images, together with the complete SHELXL structure-factor input and output files, have been deposited in the Macromolecular Xtallography Raw Data Repository (MX-RDR) with DOI https://doi.org/10.18150/D5XSNJ.

Related literature
The following references are cited in the supporting information for this article: Dong (2003)